loss of p120-catenin induces metastatic progression of ... · ofp120 resulted in anoikis...

2013;73:4937-4949. Published OnlineFirst June 3, 2013.Cancer Res Ron C.J. Schackmann, Sjoerd Klarenbeek, Eva J. Vlug, et al. Factor Receptor SignalingCancer by Inducing Anoikis Resistance and Augmenting Growth Loss of p120-Catenin Induces Metastatic Progression of Breast

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Tumor and Stem Cell Biology

Loss of p120-Catenin Induces Metastatic Progression ofBreast Cancer by Inducing Anoikis Resistance andAugmenting Growth Factor Receptor Signaling

Ron C.J. Schackmann1,2, Sjoerd Klarenbeek4, Eva J. Vlug1, Suzan Stelloo1, Miranda van Amersfoort1,Milou Tenhagen1, Tanya M. Braumuller4, Jeroen F. Vermeulen1, Petra van der Groep1,3, Ton Peeters1,Elsken van der Wall3, Paul J. van Diest1, Jos Jonkers4, and Patrick W.B. Derksen1,2

AbstractMetastatic breast cancer remains the chief cause of cancer-related death among women in the Western

world. Although loss of cell–cell adhesion is key to breast cancer progression, little is known about theunderlying mechanisms that drive tumor invasion and metastasis. Here, we show that somatic loss of p120-catenin (p120) in a conditional mouse model of noninvasive mammary carcinoma results in formation ofstromal-dense tumors that resemble human metaplastic breast cancer and metastasize to lungs and lymphnodes. Loss of p120 in anchorage-dependent breast cancer cell lines strongly promoted anoikis resistancethrough hypersensitization of growth factor receptor (GFR) signaling. Interestingly, p120 deletion alsoinduced secretion of inflammatory cytokines, a feature that likely underlies the formation of the prometa-static microenvironment in p120-negative mammary carcinomas. Our results establish a preclinical platformto develop tailored intervention regimens that target GFR signals to treat p120-negative metastatic breastcancers. Cancer Res; 73(15); 4937–49. �2013 AACR.

IntroductionAdherens junctions are required to maintain epithelial

tissue integrity. They are established by homotypic interac-tions between E-cadherin (CDH1) molecules, which in turncontrol binding of cytosolic catenins that provide linkage toand regulation of the microtubule and actin cytoskeleton (1).Loss or temporal downregulation of E-cadherin is stronglylinked to tumor development and progression of severalcancer types (2). In breast cancer, timing of adherensjunction inactivation has considerable impact on tumoretiology and cellular biochemistry. Although mutationalinactivation of E-cadherin is an initiating and causal eventin the development of invasive lobular carcinoma (ILC;refs. 3–5), late epigenetic silencing may underlie the pro-gression of invasive ductal carcinoma (IDC; ref. 6) in aprocess called epithelial-to-mesenchymal transition (EMT;

ref. 7). In conjunction with this is the finding that whiletranslocation of p120-catenin (p120; CTNND1) upon E-cad-herin inactivation controls Rock-dependent metastasis ofILC, IDC cells do not show dependency on this pathway (8,9). Thus, p120 may play context-dependent roles in thedevelopment and progression of breast cancer.

Under physiologic conditions, p120 binds to the intracellularjuxtamembrane domain of E-cadherin (10). Here, p120 con-trols E-cadherin stability and turnover in a process mediatedby Hakai (CBLL1), (11), presenilin-1 (PSEN1; ref. 12), and/orNumb (NUMB; ref. 13). Others and we have shown that geneticablation of E-cadherin or p120 in mouse mammary epithelialcells results in the induction of apoptosis, indicating thatinactivation of adherens junction function is not tolerated inthemammary gland (4, 14, 15). In contrast, genetic inactivationin other organ systems does not induce cell death, but insteadinduces impaired tissue homeostasis and hyperplasia (16–19).Also, p120 inactivation in mice seems to result in inflamma-tion, which may be caused by a loss of barrier function and theproduction of inflammatory chemoattractants (17, 18, 20).Interestingly, recent data showed that p120 can function asa bona fide tumor suppressor in the upper gastrointestinaltract. Here, somatic p120 inactivation induced the develop-ment of squamous cell carcinoma, which was accompanied byautocrine production of monocyte/macrophage attractants,thus promoting a proinvasive tumor microenvironment (21).

Several studies have indicated that p120 may be lost orinactivated in approximately 10% of IDC breast cancers cases.Loss was defined as absence of expression in more than 10%of the tumor cells, and correlated to absence of progesteronereceptor (PGR) expression and poor prognosis (22–24). Here,

Authors' Affiliations: 1Department of Pathology, 2Cancer Center, and3Division of Internal Medicine, University Medical Center Utrecht, Utrecht;and 4Division of Molecular Pathology and Cancer Systems Biology Center,The Netherlands Cancer Institute, Amsterdam, the Netherlands

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

R.C.J. Schackmann and S. Klarenbeek contributed equally to this work.

Corresponding Author: Patrick W.B. Derksen, Department of Pathology,University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, theNetherlands. Phone: 31-88-7568068; Fax: 31-30-2544990; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-13-0180

�2013 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 4937





we have analyzed p120 expression in a comprehensive set ofhuman invasive breast cancer samples and studied the con-sequences of inactivation of p120 in mammary tumor devel-opment and progression in the context of p53 (Trp53) lossusing conditional mouse models. Mammary-specific p120loss in mice resulted in a switch from noninvasive to met-astatic mammary carcinoma that phenotypically resembledhuman metaplastic breast cancer. Furthermore, inactivationof p120 resulted in anoikis resistance, which was exacerbatedby a sensitization to growth factor receptor (GFR) signalingdue to inactivation of the adherens junction. Finally, weshow that loss of p120 results in secretion of inflammatorycytokines, which may promote formation of a prometastatictumor microenvironment.

Materials and MethodsAdditional experimental procedures are described in detail

in the Supplementary Experimental Procedures.

Patient materialOf note, 298 cases of IDC were collected and histologically

examined as described in the Supplementary ExperimentalProcedures.

Antibodies and cytokine arrayThe following antibodies were used:mouse anti-p120 [West-

ern blot (WB), 1:2,000; immunofluorescence (IF), 1:500; IHC,1:500; clone 98/pp120; BD Biosciences], tetramethyl-rhoda-min-conjugated p120 (immunofluorescence, 1:150; BD Bios-ciences) mouse anti-E-cadherin (WB, 1:2,000; clone 36/E-cad-herin; BD Biosciences), fluorescein isothiocyanate (FITC)-con-jugated E-cadherin (IF, 1:150; BD Biosciences), mouse anti-E-cadherin (IHC, 1:200; clone 4A2C7; Zymed, Invitrogen), rat anti-CK 8 (Troma-1; IHC, 1:125; Developmental Studies HybridomaBank products), rabbit anti-CK14 (IHC, 1:10,000; BabCo), guin-ea pig anti-vimentin (IHC, 1:400; RDI), rabbit anti-SMA (IHC,1:350; Lab Vision), rat anti-mouse F4/80 (IHC, 1:300; Serotec),mouse anti-b-catenin (WB, 1:2,000; BD Biosciences), rabbitanti-aE-catenin (WB, 1:2,000; Sigma) goat anti-AKT1 (C-20;WB, 1:1,000; Santa Cruz Biotechnology), rabbit anti-p-AKT1s473

(WB, 1:1,000; Cell Signaling Technology), rabbit anti-ERK2 (C-14; Santa Cruz Biotechnology), rabbit anti-p-MAPK (p44/42;WB, 1:2,000; Cell Signaling Technology),mouse anti-RhoA (WB,1:250; Santa Cruz Biotechnology), mouse anti-Rac1 (WB,1:1,000; Upstate), rabbit anti-Cdc42 (WB, 1:250; Santa CruzBiotechnology), rabbit anti-EGFR (WB, 1:500; Cell SignalingTechnology), rabbit anti-p-EGFRT1068 (WB, 1:1,000; Cell Signal-ing Technology), and sheep anti-digoxigenin (Roche). Second-ary antibodies: horseradish peroxidase (HRP)–conjugated rab-bit anti-goat, goat anti-mouse, (1:2,000; Dako), HRP-conjugatedgoat anti-rabbit (1:2,000; Cell Signaling Technology), rabbit anti-sheep antibodies (Dako), Alexa Fluor 561–conjugated anti-mouse antibodies (Molecular Probes), goat anti-rat Alexa Fluor488 (Invitrogen), goat anti-rabbit Alexa Fluor 555 (Invitrogen),biotin-conjugated anti-guinea pig (Jackson ImmunoResearch).Biotin label-basedmouse antibody array was used according tothe manufacturer's recommendations (RayBiotech AAM-BLM-1-4).

Mouse crossings genotyping and generation of cell linesp120 conditional mice containing loxP sites in intron 2 and

8 of Ctnnd1, (Black-swiss;129SvEvTac; ref. 17) were a kind giftfromAl Reynolds (Vanderbilt UniversityMedical Center, Nash-ville, TN) and crossed onto the Wcre;Trp53F/F mouse model(FVB/N;Ola129/sv; refs. 4, 5). Cohorts of Wcre;Ctnnd1F/þ;Trp53F/F and Wcre;Ctnnd1F/F;Trp53F/F mice were bred fromthe resulting offspring. Wcre;Ctnnd1F/þ;Trp53F/F mice weresubsequently used to generate Wcre;Trp53F/F mice to controlfor the introduction of Black-swiss;129SvEvTac genetic mate-rial. Mice were bred andmaintained on amixed background of(FVB/N;Ola129/sv). Genotyping was done by PCR as describedpreviously (4, 17). Mice weremonitored for the development ofmammary tumors by palpation and euthanized by CO2 inha-lation when mammary tumor size reached a diameter of 10mm. Full autopsies were conducted for the analysis of tumorhistology and the detection of metastases. Age at the time ofeuthanasia was used to generate the tumor-free survivalcurves. For the generation of tumor cell lines, tumors fromWcre;Ctnnd1F/F;Trp53F/F animals were extracted, minced byhand using scalpel blades, and plated onto regular culturedishes in Dulbecco's Modified Eagle Medium (DMEM)-F12medium as described previously (5).

PlasmidsFor stable knockdown of p120, previously described

sequences were cloned into a doxycycline-inducible lentiviralexpression system (8). For p120 reconstitution experiments, alentiviral p120-1A cDNA expression construct was generatedas described in the Supplementary Experimental Procedures.

Virus production and Rho GTP pulldownLentivirus production and transductions were done as

described previously (5). In short, 106 Cos-7 cells were seededonto 10-cm Petri dishes and transiently transfected after 24hours with third-generation packaging constructs (25) and theindicated viral construct using X-tremeGENE 9 reagent(Roche). Pull-down assays for GTP-loaded RhoGTP familymembers were conducted as described previously (8, 26).

Cell cultureMouse Trp53D/D-7 (WP6) and Trp53D/D-3 (KP6) cell lines

were generated from primary tumors that developed in Wcre;Trp53F/F and K14cre;Trp53F/F female mice, respectively. Cellswere cultured as described previously (5). Human breastcancer cell lines T47D and MCF7 were cultured in DMEM-F12 (Invitrogen) containing 6% fetal calf serum, 100 IU/mLpenicillin, and 100 mg/mL streptomycin. T47D and MCF7 celllines were obtained from American Type Culture Collection.Authenticity was tested on May 16, 2012 by means of short-tandem repeat (STR) profiling (LGC Standards), after whichlarge quantities of cells were frozen separately.

Western blot analysis, immunohistochemistry, andfluorescence

Western blotting was conducted as described previously(27). For immunohistochemistry and fluorescence, tissues andcells were isolated, fixed, and stained as described in the

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Supplementary Experimental Procedures. In situ hybridiza-tion–IHC double staining experiments were carried out usinglabeled PCRproducts to identify EGFmRNAasdescribed in theSupplementary Experimental Procedures. Samples were ana-lyzed using a DeltaVision RT system (Applied Precision),equipped with a CoolSnap HQ camera and SoftWorx software.Maximum projections were taken from a stack of deconvolvedimages.

Anoikis resistance and FACS analysisAnoikis resistance was analyzed by seeding cells at a density

of 20,000 cells per well (in 500 mL) in a 24-well ultra-low clusterpolystyrene culture dish (Corning). After 4 days, cells wereharvested and resuspended in 75 mL of Annexin V buffersupplemented with Annexin V (IQ Products) and propidiumiodide (Sigma-Aldrich). The percentage of anoikis-resistant cellswas defined as the Annexin V and propidium iodide–negativepopulation analyzed on a BD FACSCalibur. To determinecellular EGF-binding ability, 400 ng of Alexa Fluor 647–conju-gated EGF (Invitrogen) was incubated with 1 � 105 trypsinizedice-cold cells in 100 mL PBS. Cells were washed with PBS toremove unbound EGF-647 and subjected to fluorescence-acti-vated cell sorting (FACS) analysis.

Growth factor stimulation assaysCells were seeded at 400,000 cells per 6-well in growth

factor–free medium for 4 hours, subsequently washed andserum-starved overnight. Next, cells were stimulated withEGF (5 ng/mL; Sigma) or HGF (25 ng/mL; R&D Systems) for10minutes. Cells were placed on ice andwashed twicewith ice-cold PBS containing Ca2þ and Mg2þ, and directly lysed in lysisbuffer.

Statistical analysesStatistical analyses were conducted using GraphPad Prism 5

(GraphPad Software). Analyses on human samples were con-ducted as described previously (28). For analysis of growthpattern and metastasis formation, Fisher exact test was used.For metastasis-free survival analysis, the log-rank test wasused. For anoikis assays, statistical significance was calculatedusing the Student t test (two-tailed), showingmeasurements ofat least three independent experiments. Error bars in allexperiments represent SD or SEM as indicated, of at leasttriplicatemeasurements.We considered P values less than 0.05as statistically significant.

Ethics statementAll animal experiments were approved by the University

Animal Experimental Committee, University Medical CenterUtrecht (Utrecht, the Netherlands). Use of anonymousor coded leftover material for scientific purposes is part ofthe standard treatment contract with patients in our hospitals.

ResultsLoss of p120 expression in invasive breast cancerTo extend previous findings on loss of p120 expression in

breast cancer, we conducted immunohistochemistry (IHC) on

a panel of 298 invasive ductal breast cancers and determinedtheir clinicopathologic variables (Supplementary Table S1).Membranous p120 was scored (absent/low vs. medium/high)in three independent tissue cores per tumor. Because wehypothesized that loss of p120 may be linked to increasedtumor progression, we defined expression as absent/low ifmore than 10% of the tumor cells were negative for p120(Supplementary Fig. S1A), as has been used for E-cadherin(29). Using these parameters, we observed that 34% of the IDCsamples showed absent/low p120 staining. Upon correlation ofp120 expression levels to clinicopathologic variables, a signif-icant association was obtained between p120 loss, high tumorgrade (P ¼ 0.007), mitotic index (MAI; P ¼ 0.002), and overallabsence of hormone receptor expression (P ¼ 0.039; Supple-mentary Table S2). Although p120 expression levels did notassociate with tumor size, lymph node status, or the otherclinicopathologic variables tested (Supplementary Table S2),these data suggest that loss of p120 expression coincides withbreast cancer aggressiveness.

Somatic inactivation of p120 results in the formation ofmetastatic mammary carcinoma

Functional inactivation of the adherens junction throughsomatic inactivation of E-cadherin is a causal event in thedevelopment of ILC (5). In E-cadherin–mutant breast cancer,p120 translocates to the cytosol where it exerts a key oncogenicrole by regulating anchorage-independent tumor growth andmetastasis (8). To study the effect of p120 loss on tumordevelopment and progression of breast cancer, we introduceda conditional p120 allele (Ctnnd1F; ref. 17) onto the Wcre;Trp53F/F (4) noninvasive mammary carcinoma model to pro-duce Wcre;Ctnnd1F/þ;Trp53F/F and Wcre;Ctnnd1F/F;Trp53F/F

mice. To correct for the differences in genetic backgroundbetween the Ctnnd1F (17) and the Wcre;Trp53F/F (FVB/N;129P2/OlaHsd) mice, a control cohort was bred using Wcre;Ctnnd1F/þ;Trp53F/F litter mates to produce Wcre;Trp53F/F.Next, females were monitored for spontaneous tumor devel-opment. In contrast to conditional inactivation of E-cadherin(4, 5), we observed that themedian tumor-free latency (T50) didnot significantly change in eitherWcre;Ctnnd1F/þ;Trp53F/F (T50¼ 223 days) orWcre;Ctnnd1F/F;Trp53F/F female mice (T50¼ 214days) when compared with Wcre;Trp53F/F females (T50 ¼ 213days; P ¼ 0.5006 and 0.5859, respectively; Fig. 1A; Table 1).Mammary tumors from Wcre;Trp53F/F females were morpho-logically typed as adenocarcinomas and carcinosarcomas, withexpansive growth patterns, dense cellular sheets, and irregularbundles of polygonal to plump spindle-shaped cells (Table 1).Tumors that developed in Wcre;Ctnnd1F/þ;Trp53F/F and Wcre;Ctnnd1F/F;Trp53F/F females showed a shift from expansiveto invasive growth as compared with Wcre;Trp53F/F animals(P ¼ 0.026 and 0.006, respectively; Table 1). In contrast tosomatic E-cadherin inactivation and the development of ILC,both heterozygous and homozygous loss of p120 resulted inmetaplastic tumor cells displaying a spindle cell morphology,often expressing vimentin with focal expression of cytokeratin(CK)8 or CK14, reminiscent of an EMT (Fig. 1B, top; Supple-mentary Table S3; Supplementary Fig. S1B). Tumors developed

Loss of p120 Leads to Metastatic Breast Cancer

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B

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Figure 1. p120 is a metastasissuppressor in mammarycarcinoma. A, conditionalmammary-specific inactivation ofp120 does not influence tumor-free latency. Kaplan–Meier tumor-free survival curves for Wcre;Trp53F/F (red line) versus Wcre;Ctnnd1F/þ;Trp53F/F (black line)versus Wcre;Ctnnd1F/F;Trp53F/F

(green line). Mice were sacrificedwhen tumors reached an averagediameter of 10 mm. NS, notsignificant. B and C, loss of p120induces a switch fromnonmetastatic to metastaticmammary carcinoma. B,histopathology of consecutivesections from mammary tumorsderived from Wcre;Trp53F/F (left),Wcre;Ctnnd1F/þ;Trp53F/F (middle),and Wcre;Ctnnd1F/F;Trp53F/F

(right). Shown are hematoxylin andeosin (H&E) staining and IHC forp120 and E-cadherin. Arrow inright panel points to a preexistenthyperplastic mammary duct. C,homozygous loss of p120 leads tometastasis. Examples of distantmetastases from Wcre;Ctnnd1F/F;Trp53F/F female animals showingdisseminated tumor cells in alymph node (left) and lungs (right).Bars, 100 mm.

Schackmann et al.






with high incidence multifocally in different mammary glandsandwere characterized by amore abundant and dense stromalmicroenvironment. Mammary tumors from Wcre;Ctnnd1F/þ;Trp53F/F females did not show LOH of the Ctnnd1 locus, astumors expressed membrane-localized p120 and E-cadherin(Fig. 1B, middle and Supplementary Table S3). Mammarytumors from Wcre;Ctnnd1F/F;Trp53F/F females showed largecells with pleomorphic nuclei, coarsely clumped chromatin,and sparse cytoplasm. As expected, all tumors from Wcre;Ctnnd1F/F;Trp53F/F female mice lacked expression of membra-nous p120 and E-cadherin (Fig. 1B, right and SupplementaryTable S3).When compared with human breast cancer, tumors that

developed inWcre;Trp53F/F mice corresponded to well-differ-entiated "luminal type" IDC, with low proliferation and expan-sive growth patterns. They predominantly express luminalCK8, showing little expression of basal CK14 (SupplementaryFig. S1B and Supplementary Table S3). Also, these tumorsshow a relatively good prognosis with infrequent formation ofdistant metastases. The highly infiltrative tumors arising inWcre;Ctnnd1F/F;Trp53F/F females corresponded to poorly dif-ferentiated "basal type" tumors. As such, they presentedphenotypic features resembling human metaplastic IDC thatare characterized by a high proliferation rate, strong nuclearatypia, and expression of CK14 (KRT14) and vimentin (VIM).In contrast to ILC, mouse and human metaplastic tumorsdisplayed decreased p120 levels and a punctate/mislocalizedE-cadherin expression pattern (Supplementary Fig. S1C andSupplementary Table S3). Human metaplastic carcinomacorresponds with poor prognosis and rapid onset of distantmetastases (30).Interestingly, and in contrast to dual somatic inactivation

of E-cadherin and p53 (4, 5), we observed abundant formationof precursor carcinoma in situ (CIS) lesions inWcre;Ctnnd1F/F;Trp53F/F females (Fig. 2A). CIS lesions showed loss of p120,but occasionally retained low levels of E-cadherin expression(Fig. 2B and C), indicating that upon p120 inactivationresidual E-cadherin remains expressed in a temporal manner,which may underlie the initial noninvasive nature of theCIS-type structures.Next, we examinedwhether the acquisition of invasive behav-

ior upon p120 ablation resulted in an increased metastatic rate.We observed a significant increase in tumor cell dissemination

in Wcre;Ctnnd1F/F;Trp53F/F versus Wcre;Ctnnd1F/þ;Trp53F/F andWcre;Trp53F/F female mice (both P < 0.05; Table 1), a phenotypenot observed in previously publishedmodels where inactivationwas targeted to the gastrointestinal tract or skin (17, 18, 20, 21).Metastases phenotypically resembled the primary tumor andlocalized to regional or distant lymph nodes and lungs (Fig. 1C).Because tumor-free latency was identical in Wcre;Ctnnd1F/þ;Trp53F/F versus Wcre;Ctnnd1F/F;Trp53F/F mice, but only Wcre;Ctnnd1F/F;Trp53F/F showed metastatic dissemination, we gen-erated primary cultures from both tumor models and assayedanoikis resistance, a hallmark of metastatic cells (5, 31). Inagreement with the in vivo metastatic behavior, we observedthat tumor cells derived from Wcre;Ctnnd1F/F;Trp53F/F femalemice (Ctnnd1D/D;Trp53D/D) were anoikis resistant, whereasneither cells derived from Wcre;Ctnnd1F/þ;Trp53F/F nor Wcre;Trp53F/Fmammary tumors (Ctnnd1D/þ;Trp53D/D and Trp53D/D,respectively) survived under these conditions (Fig. 2D). Theseresults suggested that homozygous inactivation of p120 isnecessary for the acquisition of anchorage independence andsubsequent dissemination of mammary tumor cells. In conclu-sion, we show that conditional loss of p120 induces a transitionto highly invasive and metastatic mammary carcinoma.

Loss of p120 results in loss of the adherens junction,transition to a mesenchymal phenotype, and anoikisresistance

Because we observed anoikis resistance in primary tumorcells derived from Wcre;Ctnnd1F/F;Trp53F/F female mice (Fig.2D), we examined if p120 inactivation was the causal event inthis anchorage-independent survival phenotype. To this end,we made use of Trp53D/D tumor cell lines previously generatedfrom adenocarcinomas that developed in either K14Cre;Trp53F/F (32) or Wcre;Trp53F/F female mice (4). Two indepen-dent Trp53D/D cell lines were transduced using a doxycycline-inducible lentiviral construct targeting p120 (p120-iKD). Doxy-cycline administration resulted in a strong reduction of p120protein expression (Fig. 3A), which was accompanied by adecrease in both E-cadherin and b-catenin levels and a slightdecrease in a-catenin protein expression (Supplementary Fig.S2A and S2B). Upon p120-iKD, cells lost their typical epithelialappearance and transitioned toward a mesenchymal andmotile phenotype (Supplementary Fig. S2F). To confirm thespecificity of the RNA interference (RNAi) sequence used, we

Table 1. Mammary tumor spectrum of Wcre;Trp53F/F, Wcre;Ctnnd1F/þ;Trp53F/F and Wcre;Ctnnd1F/F;Trp53F/F female mice

GenotypeNumberof mice

Medianlatency, d Metastasis

Localinvasion AC SC/CS mILC

Wcre;Trp53F/F 7 213 0 (0%) 0 (0%) 3 (43%) 7 (100%) 0 (0%)Wcre;Ctnnd1F/þ;Trp53F/F 20 223 1 (5%) 10 (50%) 1 (5%) 19 (95%) 1 (5%)Wcre;Ctnnd1F/F;Trp53F/F 21 214 9 (43%) 14 (67%) 3 (14%) 18 (86%) 1 (5%)

NOTE: If tumors were composed of two separate histologic types, both were counted separately.Abbreviations: AC, adenocarcinoma, glandular-typemammarycarcinoma;SC/CS, solid carcinoma/carcinosarcoma, tumor consistingof epithelial and mesenchymal cell types.







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Figure 2. Somatic inactivation ofp120 leads to development of CIS.A, CIS formation in premalignantWcre;Ctnnd1F/F;Trp53F/F femalemice. Shown are two separatehematoxylin and eosin (H&E)stainings. B and C, residualE-cadherin expression in CIS-typelesions in Wcre;Ctnnd1F/F;Trp53F/F

femalemice.B andC,H&Estainingand IHC for p120 and E-cadherinon consecutive sections (B) and IFstainings for E-cadherin (green)and p120 (red) in a normalmammary duct (top) and aCIS-type mammary lesion(bottom) from a Wcre;Ctnnd1F/F;Trp53F/F female mouse (C). Notethe presence of E-cadherin(arrows) in the absence of p120expression. Bars, 10 mm. D,metastatic capacity correlates toanoikis resistance. Trp53D/D cellline (white bar) and primarycultures derived from tumors thatdeveloped in Wcre;Ctnnd1F/þ;Trp53F/F (gray bars) and Wcre;Ctnnd1F/F;Trp53F/F (black bars)female mice were subjected to 4days of anchorage independentculturing and subsequent anoikisresistance analysis using FACS.�, P < 0.005. Error bars representSD of triplicate experiments.

Schackmann et al.






reconstituted cells with a nontargetable p120 cDNA to nearendogenous levels, which completely reverted the doxycycline-induced EMT phenotype (Fig. 3A and Supplementary Fig. S2Eand S2F).We next cultured two independent Trp53D/D;p120-iKD cell

lines in suspension and assayed anoikis resistance in thepresence and absence of doxycycline. Although the majorityof untreated Trp53D/D;p120-iKD cells underwent anoikis,administration of doxycycline-induced anoikis resistance (Fig.3B), which could be fully reverted upon expression of a non-

targetable p120 construct (Fig. 3B). To substantiate thesefindings, we used two E-cadherin–expressing and anchor-age-dependent human breast cancer cell lines (T47D andMCF7) inwhichwe conducted knockdown of p120 and assayedanchorage independence. MCF7 was previously reported toacquire anchorage independence in soft agar upon constitutivep120 KD (9). Indeed, as for the mouse mammary carcinomacells, p120-iKD induced downregulation of the adherens junc-tion members and functionally induced anoikis resistance(Supplementary Fig. S2C and S2D and Fig. 3C and D). Inconclusion, our data show that p120 knockdown results inloss of adherens junction function, and suggests that thisunderlies acquisition of anoikis resistance. Because thesefeatures are well-known hallmarks of malignancy (33), ourfindings suggest that loss of p120 may lead to metastasisthrough acquisition of anchorage independence.

Loss of p120 potentiates GFR signalingUpon knockdown of p120 inMCF7, cells acquire Rac-depen-

dent anchorage-independent survival, which is probably due torelieve of E-cadherin–mediated inhibition of Ras (KRAS; ref. 9).Although we could confirm that active Rac1 (RAC1) levelsincreased inMCF7uponp120-iKD,wedid not detect activationof Rac1 in either Trp53D/D;p120-iKD cell lines or human T47D;p120-iKD cells after doxycycline administration (Supplemen-tary Fig. S3). Also, no changes were observed in the levels oractivity of the other Rho family members RhoA (RHOA) andCdc42 (CDC42; Supplementary Fig. S3C), indicating that loss ofp120 in these cell systems does not result in aberrant RhoGTPase activation.

Formation or functional disruption of adherens junctionscan affect EGF receptor (EGFR) activity (34). Whether adhe-rens junction disruption results in activation or inhibition ofEGFR seems to be largely cell type–dependent (35, 36). Becausep120 knockdown resulted in a loss of adherens junctionformation through downregulation of E-cadherin, we won-dered whether GFR signaling was affected. To this end, westimulated doxycycline-treated Trp53D/D;p120-iKD cells withEGF and assayed EGFR phosphorylation. Although EGF stim-ulation in control-iKD cells induced a modest EGFR tyrosinephosphorylation, knockdown of p120 resulted in a 1.5- to 2.0-fold increased phosphorylation (Fig. 4A, compare lanes 3 and 4;quantification of three independent experiments in Fig. 4B;nonstimulated in Supplementary Fig. S5A). In line with thesefindings, we observed that the downstream PI3K/AKT andmitogen-activated protein kinases (MAPK) pathways dis-played a 3- to 5-fold increased EGF-induced phosphorylationupon knockdown of p120 (Fig. 4A and B). To substantiate thesefindings, we made use of the Ctnnd1D/D;Trp53D/D cell lines.Similarly to the GFR sensitization after inducible p120 knock-down, Ctnnd1D/D;Trp53D/D cells showed a robust increase inEGF-induced phosphorylation of EGFR and MAPK and amodest induction of phosphorylated AKT as compared withTrp53D/D cells (Supplementary Fig. S6A). Furthermore, EGFstimulation of MCF7;p120-iKD cells also showed a sensitiza-tion of EGFR, AKT, and MAPK phosphorylation upon knock-down of p120 (Fig. 4C and D). The increased EGFR signalingfollowing p120 knockdown was not due to increased EGFR

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Figure 3. Loss of p120 results in anoikis resistance of E-cadherin–expressing breast cancer cells. A, two independent Trp53D/D cell lineswere transduced with viruses carrying doxycycline (Dox)-inducible p120short hairpin RNAs (shRNA; p120-iKD) and a nontargetable p120isoform1A (p120-1A). Shown is the extent of p120 knockdown and p120-1A expression levels. Arrows indicate p120 isoforms. AKT was used as aloading control. B, Trp53D/D;Control-iKD-, p120-iKD-, and p120-1A–expressing mammary carcinoma cells were cultured in the presence orabsence of doxycycline for 4 days before subjecting cells to 4 days ofanchorage-independent culturing and subsequent anoikis resistanceanalysis using FACS. C and D, T47D (C) and MCF7 (D) were transducedwith viruses carrying p120-iKD constructs targeting human p120, treatedwith doxycycline for 4 days, and subjected to nonadherent culturing.Anoikis resistance was analyzed after 4 days. Bottom, the extent of p120knockdown (top blot). AKT was used as loading control (bottom blot).�, P < 0.005. Error bars represent SD of triplicate experiments.







levels or EGF binding at the plasma membrane (Fig. 4A andSupplementary Fig. S5C). However, we cannot exclude thatp120 knockdown induced autocrine activation of GFR signal-ing in the presence of anchorage, as serum starvation resultedin low levels of activated EGFR, AKT, and MAPK signaling(Supplementary Fig. S5A and S5B). To determine whether thesensitized GFR signaling was EGFR-specific, we also stimulat-ed the cellswith hepatocyte growth factor (HGF) to activate thereceptor tyrosine kinase MET. In line with the effects on EGF-dependent signaling, we observed a marked increase of AKTand MAPK phosphorylation upon p120 knockdown and sub-sequent HGF treatment of mouse and human cells (Supple-mentary Fig. S4A–S4C), indicating that p120-controlled GFRsignaling is a general mechanism. To validate whether theincreased GFR sensitivity also occurred in vivo, we investigatedthe expression of p-AKT and p-MAPK in tumors from Wcre;Trp53F/F;Ctnnd1F/F andWcre;Trp53F/F femalemice. Indeed, lossof p120 induced activation of AKT, and to a lesser extentMAPK

signals (Supplementary Fig. S6C). In conclusion, our data implythat loss of p120 leads to an increased sensitization of GFRsignaling in breast cancer cells.

GFR sensitization stimulates anchorage-independentviability

Because knockdown of p120 resulted in increased growthfactor sensitization of pathways implicated in growth andsurvival, we used EGF stimulation to examine whether thismechanism could increase anchorage-independent growth andsurvival of breast cancer cells. We therefore plated Trp53D/D;p120-iKD and Ctnnd1D/D;Trp53D/D cells under anchorage-inde-pendent conditions and assayed anoikis resistance. In controlcells, EGF stimulation did not mediate survival. However,addition of EGF led to an additional increase in anoikis resis-tance upon knockdown of p120 (Fig. 4E) and in Ctnnd1D/D;Trp53D/D cell lines (Supplementary Fig. S6B). Although lessprominent, EGF stimulation also induced a significant increase

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Figure 4. Loss of p120 sensitizescells to GFR-mediated anoikisresistance. A and B, p120knockdown sensitizes EGFsignaling. Two independentTrp53D/D cell lines expressingcontrol-iKD or p120-iKDconstructs were treated withdoxycycline (Dox) for 4 days,serum starved, stimulated withEGF, and subjected to Westernblot analysis using phospho-specific antibodies against EGFR(top), AKT (middle), and MAPK(bottom). Total EGFR, AKT, andMAPK were used as loadingcontrols. Quantification is shown inB. C and D, doxycycline-treated,serum-starved MCF7;p120-iKDcellswere stimulatedwith EGF andanalyzed as in A. Quantification isshown in D. E, EGF promotesanchorage-independent survivalupon p120 loss. Anoikis resistancewas analyzed in Trp53D/D;p120-iKD cells in the presence orabsence of EGF and doxycyclineas indicated. Error bars representthe SD of triplicate experiments. F,EGF promotes anchorage-independent survival upon p120loss in human breast cancer cells.Anoikis resistance of MCF7;p120-iKD cells was assayed in thepresence or absence ofdoxycycline and EGF as indicated.�, P < 0.05. Error bars in B and Drepresent SEM of at least triplicateexperiments. Error bars in E and Frepresent the SD of triplicateexperiments.

Schackmann et al.






in anoikis resistance of MCF7;p120-iKD cells (Fig. 4F). Becauseadherens junction–dependent relieve of GFR inhibition is notspecific for a given receptor and MCF7 responds more prom-inent toHGF thanEGF,wealso stimulatedMCF7;p120-iKDcellswith HGF. In line with the EGF-dependent findings in ourmouse cell lines, we observed that activation of the METreceptor induced a prominent p120-dependent increase inanoikis resistance of MCF7 (Supplementary Fig. S4E).In conclusion, we find that loss of p120 enhances anoikis

resistance through increased sensitization of GFR signaling

pathways, amechanism thatmay stimulatemetastasis in p120-negative breast cancer.

Loss of p120 leads to a proinvasive microenvironmentOn the basis of the prominent presence of stroma in the

metaplastic p120 null mammary carcinomas and our findingthat loss of p120 induces sensitization of GFR signaling, weexamined a potential source of growth factors in metastatictumors fromWcre;Ctnnd1F/F;Trp53F/F female mice. IHC indeedrevealed an abundant presence of vimentin-expressing cells

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Figure 5. Loss of p120 inducescytokine production and leadsto the development of aprometastatic tumormicroenvironment. A, p120-deficient mammary tumors arecharacterized by an abundanttumor microenvironment. Thepresence of stromal cells/macrophages in Wcre;Ctnnd1F/þ;Trp53F/F (top) andWcre;Ctnnd1F/F;Trp53F/F (bottom) mousemammary carcinomas wereanalyzed by IHC. Shown arerepresentative stainings for p120,vimentin, and F4/80. Bars, 100 mm.B, human IDC samples werestained for p120 (left) and CD68(macrophage marker; right).Shown are representativeexamples of IDC expressing highp120 (top) and low/absent p120expression (bottom). Bars, 20 mm.C, tumor-associatedmacrophages produce EGF.Wcre;Ctnnd1F/F;Trp53F/F tumors werestained for macrophages usingimmunofluorescence (green; left)and EGF mRNA using RNA in situhybridization (red; middle). DNAwas visualized using 40,6-diamidino-2-phenylindole(DAPI; blue; right). Bars, 20 mm.







surrounding p120-negative tumor cells (Fig. 5A, middle). Wenext analyzed p120-negative carcinomas for influx of macro-phages, a renowned source of EGF (37). p120-Negative tumorsfrom mouse and human samples contained a large macro-phage-dense microenvironment (Fig. 5A and B). Moreover, acombination of IHC and RNA in situ hybridization revealedthat the tumor-associatedmacrophages expressed EGFmRNA(Fig. 5C), indicating that a paracrine source of EGF is present inthe tumor microenvironment.

In the gastrointestinal tract and skin, conditional p120 lossresulted in cytokine secretion and subsequent attraction ofimmune cells (18, 20, 21). To determine whether p120 losswould also control production of inflammatory cytokines inbreast cancer, we assayed culture supernatant from Trp53D/D;p120-iKD, Ctnnd1D/D;Trp53D/D, and control Trp53D/D cellsusing an antibody-based cytokine array. Using these tools, weobserved that loss of p120 indeed increased secretion ofnumerous cytokines. Several of these factors are responsiblefor macrophage and lymphocyte attraction/maturation andstimulation of cancer-associated fibroblasts such as IL-1a(IL1A; ref. 38), IL-11 (IL11; ref. 39), and IL-12 (IL12A; ref. 40;Supplementary Table S4). These results suggest that p120-negative tumor cells are a paracrine source for the influx ofinflammatory cells and subsequent formation of a prometa-static microenvironment.

In conclusion, we show that loss of p120 results in theexcretion of cytokines that may control influx of macrophagesand other stromal cells that can promote progression of p120-negative tumor cells through the induction of sensitized GFRssignaling pathways.

Discussionp120 Loss leads to anoikis resistance and metastasis

In breast cancer, disruption of adherens junction complexformation through early mutational inactivation of E-cadherinleads to the development of ILC (3–5). Upon inactivation of E-cadherin, p120 translocates to the cytosol, where it functions asan oncogene controlling Rock-mediated anoikis resistance ofILC cells (8). In contrast to ILC, most IDC cases retain expres-sion of a functional adherens junction complex that may beinactivated through epigenetic mechanisms at later stages ofbreast cancer progression (6). Interestingly, and in contrast tothe salivary gland (17), gastrointestinal tract (20, 21), dentalenamel (16), ocular tissue (19), and skin (18), somatic inacti-vation of p120 in the mammary gland is not tolerated (15).These tissue-specific differences are further exemplified by thefact that conditional p120 ablation in the upper gastrointes-tinal tract resulted in the formation of invasive squamousesophageal carcinomas (21).

To determine a possible role for p120 during breast cancerprogression, we crossed the conditional p120 allele (41) ontothe Wcre;Trp53F/F mouse model of noninvasive breast can-cer. Using this compound mouse model, we observed that—whereas onset and incidence of primary tumor developmentwere unaffected—homozygous p120 loss resulted in meta-static disease. Interestingly, metastatic spread was notreported in other conditional p120 tumor models (17,

18, 20). Metastatic spread of tumors from Wcre;Trp53F/F andWcre;Ctnnd1F/þ;Trp53F/F female mice was a rare event.Because E-cadherin remained expressed and derivate celllines functionally displayed anchorage dependence, it indi-cated that E-cadherin expression is the rate-limiting factorpreventing initial tumor cell invasion and metastasis uponp120 loss. Supporting this assumption is our observationthat E-cadherin is temporally retained on the cell surfaceupon p120 inactivation in preinvasive CIS-type lesions fromWcre;Ctnnd1F/F;Trp53F/F mice. Also, we have shown previ-ously that low E-cadherin expression levels are sufficient toinduce anchorage dependency in anoikis-resistant mouseILC (mILC) cells (4, 5).

Interestingly, and in contrast to mutational E-cadherin loss,we observed that somatic p120 inactivation did not result inthe formation of ILC, but instead resulted in metaplasticcarcinoma. Differences may be explained by the fact thatp120 plays a key oncogenic role in ILC progression throughmyosin phosphatase Rho interacting protein MRIP-dependentregulation of the Rho, Rock, and the cytoskeleton (8, 42).Moreover, in contrast to E-cadherin inactivation, we did notobserve metastases in typical ILC dissemination sites such asbone marrow and gastrointestinal tract in the current p120conditional knockout model. Finally, dual inactivation of p120and p53 did not accelerate tumor development as seen forcombined E-cadherin and p53 loss (4, 5). These phenotypic andfunctional differences between p120 and E-cadherin suggestthat although the overall consequence is adherens junctioninactivation and metastasis, p120 loss leads to a biochemicallyand functionally different breast cancer phenotype.

p120 Loss sensitizes cells to GFR signalingHow does p120 loss regulate anoikis resistance? Our data

indicate that p120 loss results in a decrease of E-cadherin–dependent proapoptotic signals in the absence of anchorage,resulting in anoikis. In this scenario, heterozygous p120 losswill not result in metastasis, because residual membranous E-cadherin expression will lead to anoikis in cells that escapefrom the primary tumor. An additional scenario may beprovided by GFR signaling. Membrane receptors can directlyinfluence the activity of unrelated neighboring receptors (43).Depending on cellular context, E-cadherin expression mayinfluence EGFR activation (35, 36). Indeed, studies in gastriccancer showed that germline and somatic (in-frame deleteri-ous) mutations in the E-cadherin extracellular domain lead toincreased activation of EGFR signaling (44). We show here thatinactivation of the adherens junction through loss of p120 leadsto increased sensitization of GFR signaling, a phenomenonthat is likely dependent on E-cadherin turnover. Although it isstill controversial whether this mechanism is ligand depen-dent, our data indicate that the GFR hypersensitivity uponp120 loss is not caused by upregulation of EGFR expressionlevels or increased EGF binding at the cell surface (Fig. 4A andSupplementary Fig. S5C). Given our observation that E-cad-herin–dependent inhibition of GFR signaling is not specific fora given receptor, it seems unlikely that specific posttransla-tional modifications or a specific phosphatase/kinase regu-lates this process.

Schackmann et al.






Apossible scenariomay therefore be that E-cadherin elicits ageneral inhibitory effect on the recruitment of proximal acti-vating molecules and docking adaptors such as Gab1 (GAB1),Grb2 (GRB2), or Sos (SOS1) to sites of tyrosine kinase phos-phorylation (45, 46), resulting in a dampening of pathwayactivation downstream of the GFR. Alternatively, the extracel-lular domain of E-cadherin could play a role in preventingactivation of EGFR, because shedding of E-cadherin has beenimplicated in EGF-dependent activation of the PI3K/AKT andMAPK pathways and subsequent cell survival (47). Thus,although further research is needed to delineate the exactmechanism that controls the sensitization of GFR signalingupon p120 loss, we propose that increased sensitization of GFRsignaling may be caused by a relieve of direct or indirectmechanical restriction, or inhibition of ligand binding as aresult of steric hindrance between the adherens junction andGFRs.

Loss of p120 results in a prometastaticmicroenvironmentAlthough the precise nature of the influx of inflammatory

cells may depend on the model system studied, several groupsreported that p120 ablation may lead to inflammation(18, 20, 21). In line with this, we found that loss of p120 resultsin the development of stromal dense and macrophage-richmammary tumors. Furthermore, we show that loss of p120leads to secretion of several cytokines, which may control therecruitment of inflammatory cells (e.g., macrophages) thathave been implicated in inflammation-associated cancer ini-tiation and promotion (48) and are correlated with poor

prognosis in breast cancer (49, 50). Moreover, it has been wellestablished that mammary tumor cells may instigate a para-crine loop that involves the production of chemoattractantsand subsequent recruitment of EGF-producing macrophages,resulting in activation of prometastatic pathways in tumorcells (51, 52).

In closing, we propose that p120 inactivation and subse-quent E-cadherin loss is a late event in IDC tumor progres-sion. To our knowledge, there have been no reports onmutational p120 inactivation in breast cancer. Althoughsuch mutations could have easily been missed using pastsequencing techniques and the fact that p120 loss is oftenobserved in only a small percentage of the tumor, CTNND1mutations are most probably a rare event. Conversely, thiscould also indicate that p120 inactivation in IDC may becaused by epigenetic or other indirect mechanisms, as hasbeen shown for E-cadherin (53–55). This, and because of thefact that homozygous p120 inactivation in mice does notinduce the formation of mILC, leads us to hypothesize thatinactivation of p120 probably occurs during late progressionof invasive breast cancer. Although the exact mechanismthat regulates p120 inactivation remains to be resolved, ourdata show that E-cadherin–dependent inhibition of GFRsignaling is relieved upon inactivation of p120. As a conse-quence, anoikis-resistant cells are sensitized to prometa-static growth factors. Because others and we show that p120loss also facilitates concomitant formation of a prometa-static microenvironment, inactivation of p120 induces mul-tiple hallmarks of metastatic cancer. A simplified model ofour findings is presented in Fig. 6.

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Figure 6. A model for p120 as a breast cancer metastasis suppressor. A, in the presence of p120, E-cadherin is stabilized at the plasma membraneleading to the formation of adherens junctions. The adherens junction inhibits growth factor (GF)–induced GFR activation through currently unknownmechanisms. B, upon loss of p120 the adherens junction is dismantled and E-cadherin and b-catenin are degraded. As a result, anoikis resistance is induced,and subsequently enhanced by hypersensitized GFR signaling due to the relief of E-cadherin–dependent GFR inhibition. In addition, loss of p120 inducescytokine secretion, resulting in stimulation of the tumor microenvironment. This may facilitate a paracrine loop that activates sensitized GFR signaling inp120-negative tumor cells. The exact mechanism behind this increased sensitization is currently unknown, but does not seem to involve increased GFRexpression, autocrine GFR activation, or increased growth factor binding. ERK, extracellular signal–regulated kinase.







We think that our findings may have substantial clinicalramifications. In our model, GFR signaling can be enhancedindependent of GFR expression levels. Thus, our data implythat the mere presence of certain GFRs is of clinical impor-tance, provided that breast cancer cells are p120 negative.Accordingly and depending on the GFRs expressed, patientssuffering from p120-negative IDCmay be eligible for treatmentwithGFR inhibitors targeting the expressed receptors.Wehavethus uncovered a tumor-promoting role for p120 in breastcancer progression that provides an alternate rationale fortherapeutic intervention of p120-negative metastatic breastcancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R.C.J. Schackmann, J. Jonkers, P.W.B. DerksenDevelopment of methodology: R.C.J. Schackmann, T. Peeters, P.J. van Diest,P.W.B. DerksenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.C.J. Schackmann, S. Klarenbeek, E.J. Vlug, S. Stelloo,M. Tenhagen, J.F. Vermeulen, P.J. van Diest, J. Jonkers

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R.C.J. Schackmann, S. Klarenbeek, S. Stelloo, J.F.Vermeulen, P.J. van Diest, J. Jonkers, P.W.B. DerksenWriting, review, and/or revision of the manuscript: R.C.J. Schackmann, S.Klarenbeek, J.F. Vermeulen, E. van der Wall, P.J. van Diest, J. Jonkers, P.W.B.DerksenAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S. Klarenbeek, M. van Amersfoort, M.Tenhagen, T.M. Braumuller, P. van der Groep, P.W.B. DerksenStudy supervision: J. Jonkers, P.W.B. Derksen

AcknowledgmentsThe authors thank Eva Schut-Kregel for excellent technical support, Juliet

Daniel for providing the p120-1A cDNA and Al Reynolds for the p120 conditionalmice. Participants of theMMMmeeting andmembers of the Bos, Burgering, andPathology labs are acknowledged for help and fruitful discussions. The authorsalso thank the University Medical Center (UMC) Biobank.

Grant SupportThis work was supported by the UMC Cancer Center and grants from the

NetherlandsOrganization for ScientificResearch (NWO-VIDI 917.96.318) and theDutch Cancer Society (UU-KWF 2011 5230).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received January 17, 2013; revised May 14, 2013; accepted May 15, 2013;published OnlineFirst June 3, 2013.

References1. Pokutta S, Weis WI. Structure and mechanism of cadherins and

catenins in cell–cell contacts. Annu RevCell Dev Biol 2007;23:237–61.2. Jeanes A, Gottardi CJ, Yap AS. Cadherins and cancer: how does

cadherin dysfunction promote tumor progression? Oncogene 2008;27:6920–9.

3. Berx G, Cleton-Jansen AM, Nollet F, de Leeuw WJ, van de Vijver M,Cornelisse C, et al. E-cadherin is a tumour/invasion suppressorgene mutated in human lobular breast cancers. EMBO J 1995;14:6107–15.

4. Derksen PW, Braumuller TM, van der Burg E, Hornsveld M, MesmanE, Wesseling J, et al. Mammary-specific inactivation of E-cadherinand p53 impairs functional gland development and leads to pleo-morphic invasive lobular carcinoma in mice. Dis Model Mech 2011;4:347–58.

5. Derksen PW, Liu X, Saridin F, van der Gulden H, Zevenhoven J, EversB, et al. Somatic inactivation of E-cadherin and p53 in mice leads tometastatic lobular mammary carcinoma through induction of anoikisresistance and angiogenesis. Cancer Cell 2006;10:437–49.

6. Gould Rothberg BE, Bracken MB. E-cadherin immunohistochemicalexpression as a prognostic factor in infiltrating ductal carcinoma of thebreast: a systematic review and meta-analysis. Breast Cancer ResTreat 2006;100:139–48.

7. Kalluri R, Weinberg RA. The basics of epithelial–mesenchymal tran-sition. J Clin Invest 2009;119:1420–8.

8. Schackmann RC, van Amersfoort M, Haarhuis JH, Vlug EJ, Halim VA,Roodhart JM, et al. Cytosolic p120-catenin regulates growth of met-astatic lobular carcinoma throughRock1-mediated anoikis resistance.J Clin Invest 2011;121:3176–88.

9. Soto E, Yanagisawa M, Marlow LA, Copland JA, Perez EA, Ana-stasiadis PZ. p120 catenin induces opposing effects on tumor cellgrowth depending on E-cadherin expression. J Cell Biol 2008;183:737–49.

10. Reynolds AB, Daniel J, McCrea PD, Wheelock MJ, Wu J, Zhang Z.Identification of a new catenin: the tyrosine kinase substratep120cas associates with E-cadherin complexes. Mol Cell Biol 1994;14:8333–42.

11. Fujita Y, KrauseG, ScheffnerM, Zechner D, LeddyHE, Behrens J, et al.Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis ofthe E-cadherin complex. Nat Cell Biol 2002;4:222–31.

12. Baki L, Marambaud P, Efthimiopoulos S, Georgakopoulos A, Wen P,CuiW, et al. Presenilin-1 binds cytoplasmic epithelial cadherin, inhibitscadherin/p120 association, and regulates stability and function of thecadherin/catenin adhesion complex. Proc Natl Acad Sci U S A 2001;98:2381–6.

13. Sato K,Watanabe T,Wang S, KakenoM,MatsuzawaK,Matsui T, et al.Numb controls E-cadherin endocytosis through p120 catenin withaPKC. Mol Biol Cell 2011;22:3103–19.

14. Boussadia O, Kutsch S, Hierholzer A, Delmas V, Kemler R. E-cadherinis a survival factor for the lactating mouse mammary gland. Mech Dev2002;115:53–62.

15. Kurley SJ, Bierie B, Carnahan RH, Lobdell NA, Davis MA, Hofmann I,et al. p120-catenin is essential for terminal end bud function andmammary morphogenesis. Development 2012;139:1754–64.

16. Bartlett JD, Dobeck JM, TyeCE, Perez-MorenoM, StokesN,ReynoldsAB, et al. Targeted p120-catenin ablation disrupts dental enameldevelopment. PLoS ONE 2010;5:e12703.

17. Davis MA, Reynolds AB. Blocked acinar development, E-cadherinreduction, and intraepithelial neoplasia upon ablation of p120-cateninin the mouse salivary gland. Dev Cell 2006;10:21–31.

18. Perez-Moreno M, Davis MA, Wong E, Pasolli HA, Reynolds AB, FuchsE. p120-catenin mediates inflammatory responses in the skin. Cell2006;124:631–44.

19. Tian H, Sanders E, Reynolds A, van Roy F, van Hengel J. Ocularanterior segment dysgenesis upon ablation of p120 catenin in neuralcrest cells. Invest Ophthalmol Vis Sci 2012;53:5139–53.

20. Smalley-FreedWG, Efimov A, Burnett PE, Short SP, Davis MA, Gumu-cio DL, et al. p120-catenin is essential for maintenance of barrierfunction and intestinal homeostasis in mice. J Clin Invest 2010;120:1824–35.

21. Stairs DB, Bayne LJ, RhoadesB, VegaME,Waldron TJ, Kalabis J, et al.Deletion of p120-catenin results in a tumor microenvironment withinflammation and cancer that establishes it as a tumor suppressorgene. Cancer Cell 2011;19:470–83.

22. Dillon DA, D'Aquila T, Reynolds AB, Fearon ER, Rimm DL. Theexpression of p120ctn protein in breast cancer is independent ofalpha- and beta-catenin andE-cadherin. AmJPathol 1998;152:75–82.

23. Nakopoulou L, Gakiopoulou-Givalou H, Karayiannakis AJ, Gianno-poulou I, Keramopoulos A, Davaris P, et al. Abnormal alpha-catenin

Schackmann et al.






expression in invasive breast cancer correlates with poor patientsurvival. Histopathology 2002;40:536–46.

24. Talvinen K, Tuikkala J, NykanenM, Nieminen A, Anttinen J, NevalainenOS, et al. Altered expression of p120catenin predicts poor outcome ininvasive breast cancer. J Cancer Res Clin Oncol 2010;136:1377–87.

25. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, et al. Athird-generation lentivirus vector with a conditional packaging system.J Virol 1998;72:8463–71.

26. Reid T, Furuyashiki T, Ishizaki T,WatanabeG,WatanabeN,FujisawaK,et al. Rhotekin, a new putative target for Rho bearing homology to aserine/threonine kinase, PKN, and rhophilin in the rho-binding domain.J Biol Chem 1996;271:13556–60.

27. DerksenPW,Tjin E,MeijerHP,KlokMD,MacGillavryHD, vanOersMH,et al. Illegitimate WNT signaling promotes proliferation of multiplemyeloma cells. Proc Natl Acad Sci U S A 2004;101:6122–7.

28. Vermeulen JF, van de Ven RA, Ercan C, van der Groep P, van derWall E, Bult P, et al. Nuclear Kaiso expression is associated withhigh grade and triple-negative invasive breast cancer. PLoS ONE2012;7:e37864.

29. Birchmeier W, Hulsken J, Behrens J. E-cadherin as an invasionsuppressor. Ciba Found Symp 1995;189:124–36.

30. Luini A, Aguilar M, Gatti G, Fasani R, Botteri E, Brito JA, et al.Metaplastic carcinoma of the breast, an unusual disease with worseprognosis: the experience of the European Institute of Oncology andreview of the literature. Breast Cancer Res Treat 2007;101:349–53.

31. Douma S, Van Laar T, Zevenhoven J, Meuwissen R, Van Garderen E,Peeper DS. Suppression of anoikis and induction of metastasis by theneurotrophic receptor TrkB. Nature 2004;430:1034–9.

32. Liu Y, Xu HT, Dai SD, Wei Q, Yuan XM, Wang EH. Reduction of p120(ctn) isoforms 1 and 3 is significantly associated with metastaticprogression of human lung cancer. APMIS 2007;115:848–56.

33. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.Cell 2011;144:646–74.

34. Takahashi K, Suzuki K. Density-dependent inhibition of growthinvolves prevention of EGF receptor activation by E-cadherin-medi-ated cell-cell adhesion. Exp Cell Res 1996;226:214–22.

35. Qian X, Karpova T, Sheppard AM, McNally J, Lowy DR. E-cadherin-mediated adhesion inhibits ligand-dependent activation of diversereceptor tyrosine kinases. EMBO J 2004;23:1739–48.

36. ReddyP, Liu L,RenC, LindgrenP,BomanK,ShenY, et al. Formation ofE-cadherin-mediated cell-cell adhesion activates AKT and mitogenactivated protein kinase via phosphatidylinositol 3 kinase and ligand-independent activation of epidermal growth factor receptor in ovariancancer cells. Mol Endocrinol 2005;19:2564–78.

37. Pollard JW. Macrophages define the invasive microenvironment inbreast cancer. J Leukoc Biol 2008;84:623–30.

38. Nozaki S, Sledge GW Jr, Nakshatri H. Cancer cell-derived interleukin1alpha contributes to autocrine and paracrine induction of pro-met-astatic genes in breast cancer. Biochem Biophys Res Commun 2000;275:60–2.

39. Musashi M, Clark SC, Sudo T, Urdal DL, Ogawa M. Synergisticinteractions between interleukin-11 and interleukin-4 in support ofproliferation of primitive hematopoietic progenitors of mice. Blood1991;78:1448–51.

40. Trinchieri G. Interleukin-12: a cytokine at the interface of inflammationand immunity. Adv Immunol 1998;70:83–243.

41. Davis MA, Ireton RC, Reynolds AB. A core function for p120-catenin incadherin turnover. J Cell Biol 2003;163:525–34.

42. Shibata T, Kokubu A, Sekine S, Kanai Y, Hirohashi S. Cytoplasmicp120ctn regulates the invasive phenotypes of E-cadherin-deficientbreast cancer. Am J Pathol 2004;164:2269–78.

43. Chen X, Gumbiner BM. Crosstalk between different adhesion mole-cules. Curr Opin Cell Biol 2006;18:572–8.

44. Bremm A, Walch A, Fuchs M, Mages J, Duyster J, Keller G, et al.Enhanced activation of epidermal growth factor receptor caused bytumor-derived E-cadherin mutations. Cancer Res 2008;68:707–14.

45. Holgado-MadrugaM,EmletDR,MoscatelloDK,GodwinAK,WongAJ.A Grb2-associated docking protein in EGF- and insulin-receptor sig-nalling. Nature 1996;379:560–4.

46. Yamasaki S, Nishida K, Yoshida Y, Itoh M, Hibi M, Hirano T. Gab1 isrequired for EGF receptor signaling and the transformation by acti-vated ErbB2. Oncogene 2003;22:1546–56.

47. Inge LJ, Barwe SP, D'Ambrosio J, Gopal J, Lu K, Ryazantsev S, et al.Soluble E-cadherin promotes cell survival by activating epidermalgrowth factor receptor. Exp Cell Res 2011;317:838–48.

48. Balkwill F, Charles KA, Mantovani A. Smoldering and polarized inflam-mation in the initiation and promotion of malignant disease. CancerCell 2005;7:211–7.

49. Murri AM,HilmyM,Bell J,WilsonC,McNicol AM, LanniganA, et al. Therelationship between the systemic inflammatory response, tumourproliferative activity, T-lymphocytic and macrophage infiltration,microvessel density and survival in patients with primary operablebreast cancer. Br J Cancer 2008;99:1013–9.

50. Ueno T, Toi M, Saji H, Muta M, Bando H, Kuroi K, et al. Significance ofmacrophage chemoattractant protein-1 in macrophage recruitment,angiogenesis, and survival in human breast cancer. Clin Cancer Res2000;6:3282–9.

51. Condeelis J, Pollard JW.Macrophages: obligate partners for tumor cellmigration, invasion, and metastasis. Cell 2006;124:263–6.

52. Qian BZ, Pollard JW. Macrophage diversity enhances tumor progres-sion and metastasis. Cell 2010;141:39–51.

53. Batlle E, Sancho E, Franci C, Dominguez D, Monfar M, Baulida J, et al.The transcription factor snail is a repressor of E-cadherin gene expres-sion in epithelial tumour cells. Nat Cell Biol 2000;2:84–9.

54. Graff JR, Herman JG, Lapidus RG, Chopra H, Xu R, Jarrard DF, et al. E-cadherin expression is silenced by DNA hypermethylation in humanbreast and prostate carcinomas. Cancer Res 1995;55:5195–9.

55. Hajra KM, Chen DY, Fearon ER. The SLUG zinc-finger proteinrepresses E-cadherin in breast cancer. Cancer Res 2002;62:1613–8.







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